The invention relates generally to micromanipulation operations in the fields of microtechnology and nanotechnology. More specifically, the invention relates to methods and apparata for positioning micro-/nanoobjects, e.g., cells, genetic material, and molecules, in a user-defined orientation.
The micromanipulation of biological material such as cells is of extreme importance to modern biological investigation and biotechnology. Generally, a microscope, usually an inverted phase contrast microscope, is used to visualize the biological material to be manipulated. A 1, 2, or 3 axis micromanipulator is then used to manipulate and position the biological material within the view of the microscope. Usually such micromanipulators consist of a joystick and a microdrive which is moved in response to the user""s joystick actuation. Current microdrives use pneumatic, hydraulic, mechanical (e.g., screw), electromechanical (e.g., stepping motor), electromagnetic, electrostatic, piezoelectric, and magnetostrictive principles of operation to provide movement, with examples being provided in U.S. Pat. Nos. 3,835,338; 4,139,948; 4,270,838; 4,367,914; 4,610,475; 4,679,976; 4,694,230; 4,700,584; 4,749,270; 4,836,244; 4,894,579; 4,901,446; 5,325,010; 5,456,880; 5,677,709; 5,831,166; 5,845,541; 5,973,471; and 6,055,859.
Microtools, such as glass micropipettes or microcapillaries, are attached to the microdrives. The size, shape, and other characteristics of the microtool depend on the operations to be performed; for example, a micropipette for fixing (holding) oocytes generally has a round diameter of about 50-120 microns, whereas the diameter of a micropipette for fixing other cells could be less than 50 microns, or as great as 120 microns (or sometimes greater). Two or more microtools are often used simultaneously on the same microdrive. One microtool (generally a micropipette), termed the holder and denoted by the reference numeral 12 in FIG. 1, is used to fix/grasp the biological material (e.g., an oocyte) so as to effect its micromanipulation. The fixing of an oocyte 100 at the tip of the holder 12 involves supplying negative medium pressure inside the holder 12. The negative pressure can be supplied by several sources, such as a microsyringe or a vacuum device connected to the holder 12 by tubing. A vacuum device, such as in U.S. Pat. No. 5,456,880, is preferred because it provides a desired precise amount of pressure, and constant negative or positive pressure can be maintained even if medium leakage owing to non-tight contact with the oocyte at the holder tip occurs.
In some applications, precise manipulation of biological material can be of critical importance. For example, operations on oocytesxe2x80x94such as removal of genetic material from an oocyte, transferring somatic cells under an oocyte""s zona pellucida in cloning technology, and injection of genetic material into an oocyte""s pronucleus for production of transgenic animalsxe2x80x94all present problems of oocyte orientation. Oocytes must also be carefully manipulated into a precise orientation when performing IVF (in vitro fertilization) or other assisted reproduction operations such as ICSI (intracytoplasmic sperm injection), PZD or ZD (partial or full zona dissection), SUZI (sperm under zona injection). In most species, an oocyte is comprised of an animal and vegetal component, and the location of the oocyte""s first polar body identifies the animal pole. For example, referring to FIG. 1, ICSI into an oocyte 100 involves orientation of the matured oocyte 100 with the holder 12 to situate the polar body 102 at a 6 o""clock or 12 o""clock position. Otherwise, a micropipette or other microtool acting on the oocyte 100 from at or near the horizontal plane could damage the meiotic spindle and metaphase plate during ICSI.
The micromanipulation of biological cells as described in the above-referenced patents uses a combination of weakly-controlled cell rotation and translational cell motion, and generally only allows for partial (i.e., less than 360xc2x0) rotation. Usually, after being coarsely oriented in generally the desired orientation, the oocyte 100 is non-firmly fixed in the holder 12 so that it may be finely oriented by the use of other micropipettes. Finally, once the oocyte 100 is positioned as desired, it is firmly fixed on the holder 12 so further microoperations may be performed e.g., injection of spermatazoa in ICSI. Often, difficulties are encountered because the non-spherical shape of the oocyte 100 leads to undesired oocyte movement and orientation, particularly when fine orientation is being performed. Additional problems occur because the injected cells tend to stick to the surfaces of the holder 12 and.or other microtools, especially when somatic cells or spermatozoa are inside the micropipette or other injection apparatus. Thus, orientation of oocytes is often a repetitive, time-consuming, trial-and-error process, and leads to significantly decreased efficiency in operations such as cloning, IVF and the like. Time is wasted in attaining the proper orientation, and the oocyte 100 is meanwhile experiencing time damage because it is resting in a non-native environment. This is a significant factor in causing the failure of such operations.
Apart from orientation problems in biological fields, similar problems exist in the field of scanning probe microscopy (SPM) and in nanotechnology. In such fields, it is necessary to rotate and otherwise orient microparticles, nanoparticles and molecules in order to observe or manipulate them, and thereby create nanostructures and nanodevices. See, for example, U.S. Pat. Nos. 5,606,162 and 5,760,300.
The invention, which is defined by the claims set forth at the end of this document, is directed to methods and apparata for manipulating micro-/nanoobjects which at least partially alleviate the aforementioned problems. A basic understanding of some of the preferred features of the invention can be attained from a review of the following brief summary of the invention, with more details being provided elsewhere in this document.
The invention involves methods and apparata for manipulating micro-/nanoobjects wherein a microtool exerts an attractive force on the micro-/nanoobject, and a vibrator coupled to the microtool generates oscillating motion in the microtool (and more preferably orbital motion in the microtool) in at least one plane. It has been found that so long as the attractive force on the micro-/nanoobject is not too high, the oscillating motion of the microtool will cause rotation of the micro-/nanoobject, with the xe2x80x9cweakxe2x80x9d attractive force of the microtool maintaining the micro-/nanoobject adjacent the microtool during such rotation. In essence, the microtool oscillation drives the rotation of the micro-/nanoobject, while at the same time it helps to avoid sticking of the micro-/nanoobject to the microtool. Once the micro-/nanoobject has been rotated to a desired orientation, the attractive force of the microtool on the micro-/nanoobject can be increased to such a level that the micro-/nanoobject will be firmly fixed to the microtool. Such fixation is more easily accomplished if the orbital motion of the microtool is ceased after the micro-/nanoobject is rotated to the desired orientation.
Orbital motion of the microtool can be achieved by providing linear oscillating inputs to the microtool in two different directions (e.g., in two different orthogonal directions), and providing a phase difference in the oscillations. In effect, the oscillating inputs to the microtool are tailored to have the microtool move in a Lissajous pattern. Orbital motion may occur in one or more planes, i.e., in one or more degrees of freedom, to rotate micro-/nanoobjects in these planes. As an example, if the vibrator includes three oscillators affixed to the microtool which respectively cause microtool oscillation in the X, Y, and Z directions, coordination of the oscillations in the X and Y directions can generate orbital motion in the XY plane (i.e., rotation about the Z axis); coordination of the oscillations in the X and Z directions can generate orbital motion in the XZ plane (i.e., rotation about the Y axis); and coordination of the oscillations in the Y and Z directions can generate orbital motion in the YZ plane (i.e., rotation about the X axis). Rotation in these planes may occur simultaneously or sequentially. Varying the frequency, amplitude, and form (e.g., phase relationship) of the orbital motion, as well as the degree of attractive force provided by the microtool, allows the user to vary the direction and frequency (speed) of rotation of the micro/nanoobject as desired.
The microtool""s attractive force may be created by a force generator coupled to the microtool, and the force generator may take a variety of forms. A first example is a vacuum device which varies the fluid pressure inside a fluid-varying microtool such as a micropipette with respect to the ambient pressure of the fluid surrounding the micro-/nanoobject, thereby generating an attractive suction force. A second example is a charge generator which positively or negatively charges the microtool, thereby making it attractive to negatively or positively charged or polarized micro-/nanoobjects (or portions thereof). Another example is an electromagnet or similar device which magnetically polarizes the microtool, thereby making it attractive to ferromagnetic micro-/nanoobjects (or portions thereof). A final example is a device which varies the size, shape, or other characteristics of the microtool so as to generate varying degrees of Van der Waals forces in the microtool (e.g., by extending or retracting attractive filaments from the tip or other portion of the microtool).
The microtool is preferably positioned adjacent to an operating stage whereupon the micro-/nanoobjects may rest, with a microscope being positioned adjacent the microtool and the operating stage so that the user may monitor the positioning of the micro-/nanoobject and modify it as desired. While viewing the positioning of the micro-/nanoobject, the user may actuate an input device, preferably a trackball, to which the vibrator is responsive to thereby provide the user""s indicated motion inputs as output motion at the microtool. Thus, in accordance with the user""s rotational input at the trackball, corresponding rotation of the micro-/nanoobject occurs. The invention may also incorporate a microdrive which is coupled to the microtool and which may translate the microtool in one or more directions, so that the invention may allow the user to both rotate and translate the micro-/nanoobject in question.
Unlike prior manipulation inventions known to the inventor, the invention allows for rotation of a micro/nanoobject in as many as three degrees of freedom, and overall positioning of a micro/nanoobject in as many as six degrees of freedom if the invention incorporates a microdrive or other translational positioner. The invention is believed to have particular value in biotechnology applications since it decreases micromanipulation time and therefore decreases cell damage and increases cell viability. By use of the invention, the cell need not rest in non-native conditions for a long period before the cell can be properly positioned for performance of the desired microoperation. Applications where the invention proves particularly helpful include microsurgery of living cells, ICSI (intracytoplasmic sperm injection), IVF (in vitro fertilization), injection of genetic material for obtaining of transgenic animals, nuclear transfer for cloning, embryology, intracellular electrophysiology investigations, ultra-microanalysis, other fields of biotechnology, and the assembly and observation of microparticles, nanoparticles and molecules. The invention has been found to be particularly useful in the micromanipulation of oocytes and embryos.
Further advantages and objects of the invention will be apparent from the following detailed description of a preferred embodiment of the invention, which is made in conjunction with the accompanying drawings.